1. Field of the Invention
The present invention relates to enzyme immunoassay techniques for clinical or home detection of microorganisms and Neisseria gonorrohoea. The invention further relates to the use of specimen collection media and chromogenic substances for the enzyme immunoassay.
2. Description of the Prior Art
Patent application U.S. Ser. No. 410,157 filed Aug. 20, 1982 entitled "Colorimetric Detection of Bindable Substances" by Bego Gerber, Elliott Block, Izak Bahar, Mary Coseo, Cheryl Eaton, Wendy Jones, Patricia Kovac, and John Bruins commonly assigned with the present application contains related and common subject matter.
There are a number of immunoassay techniques in contemporary use for laboratory detection and measurement of antigens or antibodies present within a test sample. Most of these techniques, however, are unsuitable for use outside a laboratory setting because of complexity of the detection equipment and other difficulties inherent in conducting many conventional immunoassay techniques. Thus, there is a need for simple, reliable techniques for use in a clinical setting by a physician or a clinician or which may be readily used by a patient in a home diagnostic immunoassay kit for detection of certain diseases or conditions. It may be desired, for example, to design the immunoassay test kit to detect diseases such as gonorrhea (gonococcus antigen), conditions such as pregnancy (HCG immunogen), exact time of ovulation in women (luteinizing hormone antigen), or any one of a number of specific bacteria and other microorganisms. In order for a diagnostic immunoassay kit to be satisfactory for home use, the kit must be relatively inexpensive, the immunoassay method must be easy to use, reliable, and efficient, and above all must be safe. Additionally, the test method must be of sufficient sensitivity to easily detect the desired antigen in the test sample.
The earliest conventional immunoassay methods for detection and measurement of antigens or antibodies in a test sample are the radioimmunoassay method (RIA) and the fluorescent immunoassay technique (FIA). In the radioimmunoassay method, the antigen or antibody to be detected is either directly or indirectly labelled with radioactive isotope, commonly an isotope of iodine. Although a radioimmunoassay generally exhibits a high degree of sensitivity even for detection of trace amounts of test antigen or antibody, these tests all involve the use of hazardous radioactive materials which require special handling, storage, and disposal. Also, expensive analytical equipment is required, particularly in radioimmunoassay methods which involve the precipitation of immune complexes, which requires detailed analytical recovery techniques. Solid phase radioimmunoassays circumvent the need for detailed analytical recovery required in fhe precipitation method, but require much longer incubation times, typically between 30 to 60 hours. Therefore, in view of the potential hazard involved in handling radioactive material, and the need for expensive detection equipment and long incubation times, the radioimmunoassay method is unsuitable for application to home diagnostic kits.
In the immunofluorescence assay the test antigen or antibody may be labelled directly or indirectly by use of fluorescent dyes (fluorochromes) such as fluorscein and rhodamine which can be coupled to the test antigen or antibodies or their immunocomplexes without destroying their specificity. Such conjugates labelled with fluorescent dye can be visualized in a fluorescence microscope. Major disadvantages of the immunofluorescence method either by the direct or indirect method are firstly the dependence on expensive fluorescence microscopes for detecting the labelled conjugate, and secondly, the acknowledged difficulty in quantifying the test antibody or antigen present in the sample. Therefore, the immunofluorescence assay method is unsuitable for use in connection with home diagnostic kits.
In recent years, the enzyme immunoassay method has received increasing attention from researchers for use in detecting and measuring antibodies or antigen in test samples. The enzyme immunoassay methods involve enzyme labelling of the test antigen or antibody either directly or indirectly by labelling immunocomplexes which bind specifically to the test antigen or antibody and which catalyze reaction with a substrate. Some means is provided for monitoring enzyme activity. For example, in the measurement of the enzyme activity of oxidoreductases, one might monitor the oxidation of a chromogenic substance by a substrate such as hydrogen peroxide. Such so-called colorometric assays are readily adapted to the hometesting environment. When the chromogenic substance oxidizes, it forms a chromophore which exhibits visually discernable color changes.
Typical enzyme immunoassays include competitive EIA for antigens, and an enzyme linked immunosorbent assay (ELISA) which also includes direct and indirect ELISA methods. In the competitive EIA method, antigen labelled with enzyme competes with unlabelled sample antigen for binding to a limited quantity of antibodies which have been adsorbed onto a support medium. Once the amount of bound enzyme labelled antigen has been determined, the amount of sample antigen can be determined by the difference between the total amount of antibody bound to antigen less the amount of antibody bound to labelled antigen.
In enzyme immunoassay methods as known in the prior art, antibodies specific to the test antigen may be first adsorbed in excess amount onto a solid surface such as a plastic well or tube. The test solution containing antigen is then added; the antigen will bind to the adsorbed antibody. The solid phase, that is the phase composed of all material bound to the antibody, is then thoroughly washed to separate unbound components. Further steps are directed toward quantifying the bound antigen. In the double sandwich antibody ELISA method, an enzyme labelled second antibody, preferably having binding sites different from those of the first antibody, is added and reacts with specific determinant sites on the bound antigen. The enzyme labelled second antibody is added in excess to assure that all the antigen present in the solid phase that is bound to the first antibody will also be bound to enzyme labelled second antibody. The enzyme labelled second antibody molecules will bind in a fixed ratio to each antigen molecule depending on the valence, i.e. specific available binding sites, of the antigen for the second antibody. The solid phase is again washed to remove excess second antibody and any other unbound constituents. An enzyme substrate is then added in solution in excess amount, whereby it makes contact with the bound solid phase. For the enzyme horseradish peroxidase, the substrate may typically be composed of a solution of hydrogen peroxide and a chromogenic material.
o-Phenylenediamine (OPD) heretofore has been acknowledged as one of the most sensitive chromogenic substrates available for detection of peroxidase activity. However, OPD produces a yellowish/orange chromophore which although discernible to the unaided eye is nonetheless not a preferred color for a chromophore, since the eye is more sensitive to other colors in the light spectrum such as blue. Other conventional chromogenic compounds having good sensitivity for peroxidase enzyme detection in enzyme linked immunoassays are o-tolidine and ABTS [2,2'-azinodi(3-ethylbenzothiazolinesulfone-6) diammonium salt]. Although o-tolidine and ABTS have been used successfully for detection of a number of specific antigens using ELISA methods, these latter two chromogenic compounds each have less sensitivity than OPD. All of these chromogenic compounds have been reported as soluble and initially colorless, yielding color change upon oxidation with hydrogen peroxide. Typical enzymes that have been used in the enzyme immunoassay methods are horseradish peroxidase, glucose oxidase, .beta.-D-galactosidase, and alkaline phosphatase. However, since the latter two are found in normal human urine, they are not preferred for use in connection with enzyme immunoassay techniques if they are to be applied in home diagnostic kits. The amount of test antigen present in the solid phase of the double sandwich ELISA method is then directly measurable after the chromogenic substrate has been added, since when there is excess substrate the rate of color change of the chromogen is independent of the substrate concentration and is a function of the total enzyme concentration. The enzyme concentration is a function of the amount of enzyme labelled second antibody, which in turn is a function of the amount of test antigen. Therefore, the rate of color change is a function of the amount of test antigen. The rate of color change can be measured by means of a spectrophotometer if quantification of the amount of test antigen is desired. For use in home diagnostic kits when quantification is not required, the assay should be capable of permitting the user to detect a color change visually which in turn would indicate the presence of a specific antigen in the test sample.
In testing specifically for gonorrhea (Neisseria gonorrhoea) the fluorescence immunoassay method, or radioimmunoassay methods are not normally employed since these methods when applied to detection of gonococcus bacteria have been reported to be unreliable or else require the use of expensive microscopes or radiation detection equipment. Such equipment can only be used effectively by highly trained specialists.
Enzyme immunoassay methods have heretofore not been used successfully in diagnosis of gonorrhea because they have lacked sufficient sensitivity to detect the presence of low concentrations of gonococcus often present in infected male or female patients. The lack of sensitivity has been attributed in part to the unavailability of suitable collection medium to adequately desorb the GC cells from the collection swab and simultaneously preserve or increase the antigenic binding sites, and in part due to the unavailability of chromogen of sufficient sensitivity.
In attempts to apply enzyme immunoassay methods to detection of gonorrhea, phosphate buffered saline solutions (PBS) have typically been tried as a collection medium for desorption of gonococcus cells from clinical swabs because of the known preserving effect of this solution on gonococcus antigenic binding sites. However, a collection medium composed of PBS solution does not quickly desorb gonococcus cells from the swab, and only preserves the antigenic binding sites without increasing the total number of exposed antigenic binding sites per cell.
The standard techniques used in testing for gonorrhea have preferably been either the direct Gram staining smear technique which is a preferred technique for males, and culture methods employing a standard agar culture medium for testing women patients.
Although the Gram stained smear technique for testing gonorrhea in male patients by staining a sample urethral exudate with Gram dyes is both quick and inexpensive, it nevertheless is not recognized as a legal and definitive test. Corroboration of the results from Gram staining is typically obtained through culture analysis of the sample. This requires that a culture of the specimen be harvested in an agar medium, such as that supplied in standard Transgrow bottles or through use of standard Thayer-Martin culture medium. These tests must be performed in a CO.sub.2 rich environment and cannot be done quickly, but rather require at least about a day or longer incubation time. As a result, the culture techniques are far more time consuming and difficult to perform and are therefore not suited for home diagnostic or quick clinical evaluation. Although a positive from Gram staining of a male smear sample is a basis for diagnosis of gonorrhea subject to corroboration by the culture method, a negative result from Gram staining does not necessarily mean that the patient is free of the disease. In situations where the smear technique gives a negative result, it is particularly prudent that the test be repeated through culture by inoculating a sample specimen on a Thayer-Martin or Transgrow culture medium. Some medical experts require that yet a third test, namely a sugar fermentation test be made in order to absolutely confirm presence or absence of the disease in male patients.
The difficulty in reliably diagnosing gonorrhea in female patients is even greater than diagnosing the disease in male patients. In female patients the use of smear staining by Gram's technique is not a recommended procedure. Rather the preferred method is through culture wherein vaginal or cervical specimens are inoculated on standard agar culture media such as the Transgrow or Thayer-Martin media. The culture techniques as above-described are time consuming in that at least one day's time is typically required to harvest the culture before it is possible to make a diagnosis. Additionally, it is well recognized that the conventional Transgrow or Thayer-Martin culture techniques are not suitable for home diagnostic use but are best performed and monitored in a laboratory by skilled clinicians. Furthermore, it is recognized that diagnosis of the disease can be easily missed in women through the use of the culture methods, since endocervical specimens of infected female patients may have very low concentrations of gonococcus cells particularly during the early stages of the disease. It is well recognized that the culture techniques are unreliable for positively diagnosing the presence of gonorrhea in women during the early stages or latency period of the disease which may span a considerable period of time, from days or even weeks from date of initial infection.
Thus, there is presently no one reliable test available for diagnosing gonorrhea in male and female patients which could be performed in a home diagnostic setting. The tests which are conventionally employed require that they be carried out by skilled clinicians within a laboratory setting, particularly if the test involves the growth of culture in standard culture media, or if the test requires complex microscopic examination as in fluorescence immunoassay methods. Additionally the standard laboratory procedures, particularly those employing culture techniques, require high degree of care in maintaining the sterility of the sample and reagents. The Gram staining smear technique preferred for testing male patients, while a quick and convenient test, unfortunately does not provide the degree of reliability required of a home diagnostic test, since Gram staining may result in a negative and yet the patient could very well be infected. Also, the Gram staining technique has the serious practical disadvantage for application to home diagnostic testing in that additional instrumentation such as a microscope with specialized components is required to determine the results: Enzyme immunoassay methods or other serologic methods for diagnosting gonorrhea have been attempted but have heretofore not been employed successfully since results therefrom can be inconclusive, and such tests are unsuitable for home diagnostic study.
Conventional chromogenic reagents preferably are prepared in fresh batches just prior to use, and tend to oxidize and become colored spontaneously when left in storage, typically even for as little as one hour. In general, a chromogenic compound for detection of enzymes such as horseradish peroxidase should be relatively inexpensive, easy to use in connection with home diagnostic assays, and above all, noncarcinogenic and safe. The chromogenic compound importantly should be stable, soluble, and exhibit rapid color change upon reaction. Also, with substrate, e.g., hydrogen peroxide when exposed to oxidative enzymes, the product chromophore should likewise be safe, stable, and exhibit a high molar absorptivity.
Other chromogenic compounds have been used in pathological studies or assays outside the realm of enzyme immunoassay methods. For example, benzidine has been used to determine peroxidase activity of heme proteins. In such an application, benzidine-hydrogen peroxide chromogenic substrates have been used in forensic medicine for the detection of blood using the peroxidase activity of hemoglobin. Also, benzidine staining procedures have been used to detect the peroxidase activity of the heme proteins cytochrome P-450 and cytochrome P-420. Specifically, the peroxidase activity of these cytochromes has been detected on sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis by a benzidine staining procedure. Problems have been encountered, however, with the use of benzidine, one of the more important being that it has been found to be a potent human bladder carcinogen. Additionally, staining with benzidine may lack sensitivity. The stain exhibits limited stability, therefore making it difficult to photograph. Researchers in the field of forensic medicine have, therefore, sought alternatives to benzidine for the detection of peroxidase activity of heme proteins, in particular for detection of peroxidase activity of hemoglobin. One such alternative reported in the literature is the use of 3,3',5,5'-tetramethylbenzidine in hydrogen peroxide as a stain for the peroxidase activity of heme proteins, particulary cytochrome P-450. The results of the improved staining procedures using tetramethylbenzidine are reported in P. Thomas, B. Ryan, and W. Levin, Analytical Biochemistry 75, 168-176 (1976).
The advantages of using tetramethylbenzidine for the heme staining of cytochrome P-450 as reported in this reference were that the TMB substrates exhibited increased sensitivity, clear dull background, thereby improving color contrast, and greater staining stability, i.e., the TMB stained gels could be stored in the dark at room temperature for at least one month with only minimal loss in TMB stain intensity. In Thomas et al. supra., the improvement in stability of the TMB-hydrogen peroxide staining was reported to be in marked contrast to that obtained with benzidine-hydrogen peroxide where much of the stain is lost within one hour after heme staining for detection of cytochrome P-450. The TMB stained gels were reported to give distinct color even after 25 hours, in contrast to the results obtained with benzidine heme staining wherein much of the color was lost in only one hour after staining.
In the reference Thomas et al. supra., the preferred preparation of the TMB chromogenic solution for detecting the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate (SDS)-polyacrylamide gel was described as follows: A 6.3 mM TMB solution was freshly prepared in methanol. Immediately before use, 3 parts of the TMB solution were mixed with 7 parts of 0.25 M sodium acetate buffer (pH 5.0). After 1 or 2 hours with occasional mixing (every 10-15 min.), H.sub.2 O.sub.2 was added to a final concentration of 30 mM. The staining was visible within 3 minutes when using this solution. After the gels were stained, they were placed in an acetate buffered 30 percent isopropanol solution (i.e. the gels were placed in isopropanol: 0.25 M sodium acetate, pH 5.0 at a ratio of 3:7). This served to clear the gel background and enhance staining intensity and permitted storage of the stained gels in the dark at room temperature for at least 2 months with minimal loss in stain intensity.
The use of alternative solvents for TMB such as ethanol or isopropanol instead of methanol reportedly resulted in diminished stain intensity. The 3:7 ratio of methanol to sodium acetate buffer of pH 5.0 was found to be optimal. When the pH 5.0 sodium acetate buffer was replaced with buffers at pH 4.0, 4.5, 5.5 or 6.0, the stability and sensitivity of staining were significantly reduced.
A salt of tetramethylbenzidine, believed to be noncarcinogenic, namely tetramethylbenzidine dihydrochloride (TMB-d) has been reported in the literature and used as a suitable substitute for benzidine for staining of hemoglobin containing cells and for quantitative determination of hemoglobin in solutions. See, H. H. Liem, et al. Analytical Biochemistry, 98, 388-393 (1979). Unlike tetramethylbenzidine, TMB-d is water soluble obviating use of an organic solvent. It also dissolves in 10 percent acetic acid forming a green oxidation product. It has the disadvantage, however, of being unstable in the presence of moisture and air, thereby diminishing its effectiveness.
The use of tetramethylbenzidine-hydrogen peroxide chromogenic substrates as stains for detection of peroxidase activity of heme proteins, such as cytochromes, as reported in Thomas et al., or hemoglobin as reported in Liem is directed to the forensic sciences for detection of blood particularly in samples produced during criminal or accident investigations. The uses of tetramethylbenzidine disclosed in these references, including heme staining and staining of cytochrome or hemoglobulin for application to the forensic sciences, do not extend to the use as a chromogen in enzyme immunoassays.
Accordingly, it is an object of the present invention to provide an enzyme immunoassay test which is reliable for detection of microorganisms, antigens derived from microorganisms, and antibodies directed against the microorganisms.
It is an important object of the present invention to provide an enzyme immunoassay technique for gonorrhoea which may be performed by the patient in a home diagnostic study.
It is another important object of the invention to provide an improved collection medium for the test specimen. A related object of the invention is to provide an enzyme immunoassay test for gonorrhoea incorporating both an improved collection medium and improved chromogenic substrate.